Refinements developed during five large scale preparations of multiple complement components have allowed us to improve resolution and functional recovery of C3 (96%), C4 (57%) and C5 (65%) and to maintain C9 yield. A second stage DEAE-Sephacel column was used in which certain components (P, I, C2, B, C7, C8, and C6) obtained now as complex pools are rechromatographed under more favorable conditions. This step generated relatively pure components more amenable to purification. C1-In has been purified to homogeneity. C2 and C4 are in final stages of purification and further purification of C6 is underway. A C3 deficient strain of dogs was recently identified. To study this C3 deficiency, methods to isolate C3 from normal dogs were developed and resulted in the preparation of pure C3 determined to have a M.W. of 180,000. Further work has resulted in methods to produce and recover highly purified C5a from citrated plasma. This C5a migrates as a 15,000 dalton band on SDS-PAGE. Staining with antibody to C5 immunoblots of C5a on nitrocellulose confirm the presence of this biologically active peptide. Studies on cell-bound C4b suggest that the alpha-chain as well as the beta-chain may be important in generation C3 convertase. Lysis of nucleated cells by complement demonstrated a cooperative action of terminal complement components in contrast to one-hit kinetics for small marker release. This divergence can be interpreted as a multi-channel requirement for killing these nucleated cells.